Recombinant anti ki 67 antibody

Proliferation and apoptosis in the tumor nodules and organs were detected with IHC stainings for Ki-67 and cleaved caspase-3, respectively. Sections of 4 µm were cut, deparaffinized and heat-induced antigen retrieval was performed with 1x Sodium Citrate pH 6 at 98°C, followed by peroxidase blocking. Slides were incubated with the primary antibody Recombinant Anti-Ki-67 antibody 1:200 (anti-rabbit, Abcam) or cleaved caspase-3 1:200 (anti-rabbit, Cell Signaling) overnight at room temperature. The next day, sections were incubated with the secondary antibody Poly-HRP-anti-Rabbit IgG (Immunologic) for 60 minutes at room temperature. Afterwards, the sections were stained with PowerDAB (Immunologic), counterstained with hematoxylin (Fluka) and mounted with Pertex. Representative pictures were taken with a light microscope (Olympus) at a magnification of 40x. Percentage positive staining was analyzed using the software program QuPath version 0.2.3.

To assess changes in proliferation or apoptosis in the cells after treatment, IHC stainings for Ki-67 and cleaved caspase-3 were performed. Cells were seeded on sterile cover slips (21x26 mm) placed in 60 mm cell culture dishes and were incubated overnight at 37°C. Cells were treated as described above. After treatment, medium was refreshed and cells were incubated for 48 hours. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 minutes. After washing the cells three times with PBS, cells were permeabilized with PBS containing 0.1% Triton X-100 and 1% fetal goat serum (TNBS) for 30 minutes. Slides were incubated with the primary antibody recombinant anti-Ki67 antibody 1:200 (anti-rabbit, Abcam) or cleaved caspase-3 antibody 1:100 (anti-rabbit, Cell Signaling) for 1 hour at room temperature. After washing the cells three times with PBS slides were incubated with secondary antibody goat-anti-mouse Cy3 (Jackson ImmunoResearch) diluted 1:100 in TNBS for 30 minutes in the dark. Cells were washed twice with PBS and nuclei were stained with DAPI (2.5 µg/ml) for 20 minutes. Coverslips embedded in vectashield were sealed on microscope slides. Digital image analysis was performed using Leica LAS-X software (Leica). Pictures of at least 150 cells from three independent experiments were obtained using Leica DM6 microscope quipped with a CCD camera.

Doxorubicin was purchased from Merck (Doxorubicin, Hydrochloride - CAS 25316-40-9-Calbiochem, Merck, Germany). Hydroxyapatite powder was purchased from FLUIDINOVA, Portugal (micro hydroxyapatite powder, 10 ​μm and nano hydroxyapatite paste, <50 ​nm particle size). RPMI 1640 Medium and GlutaMAX™ Supplement were purchased from Thermo scientific, U.S.A. Heat inactivated fetal bovine serum (FBS) and MTT reagent was purchased from Sigma Aldrich, Germany. All the chemicals used in immunohistochemistry staining were purchased from Abcam: Citrate Buffer pH 6.0 (ab93678), Hydrogen Peroxide Blocking Reagent (ab64216), Protein Block (ab64226), Recombinant Anti-Ki67 antibody (ab16667), Antibody Diluent (ab64211), Goat Anti-Rabbit IgG H&L (HRP) (ab205718), DAB Substrate Kit (ab64238) and Hematoxylin Solution (Mayer's, Modified) (ab220365). TUNEL Assay Kit (ab206386) was also purchased from Abcam. ATPlite Luminescence Assay (6016943) was bought from PerkinElmer. Endocytosis inhibitors (Amiloride, LY2940002, MβCD, Dynasore) were purchased from Thermo Fisher. Hochst 33342, GFP-LAMP1, Mitotracker and Tom20 were all purchased from Thermo Fisher. 143B human osteosarcoma cells were purchased from American Type Culture Collection (ATCC). Athymic nude mice (Fox1^nu/nu) were procured from Janvier Labs (France).